Along with AFP (α-fetoprotein), PIVKA-II (Protein Induced by Vitamin K Absence or Antagonist-II) is measured widely in clinical examination laboratories as a hepatic cell tumor detecting marker which specifically increases in hepatic cell cancer patients. Generally, magnetic beads, glass beads, plastic plates, latexes or the like on which PIVKA-II specific monoclonal or polyclonal antibodies are adsorbed are subjected to a first reaction with serum or plasma. Then, after washing the reaction mixture for B/F separation, a second reaction is carried out by adding human prothrombin specific polyclonal or monoclonal antibodies labeled with an enzyme, a fluorescent material, a radioisotope, an Ru complex or the like. Then, after washing the reaction mixture for B/F separation, the absorbance or luminescence of the enzyme, the fluorescent material, the radioisotope or the Ru bound to an immune complex formed by the antigen-antibody reaction is measured to determine PIVKA-II in the serum or the plasma.
Heretofore, PIVKA-Il has been measured by an enzyme immunoassay (EIA), but the EIA has poor sensitivity with a low positive rate for a relatively small hepatoma. Accordingly, an electrochemiluminescence immunoassay (ECLIA) utilizing an antigen or an antibody which is labeled with an Ru complex was recently developed for a further highly sensitive measurement. The application of the electrochemiluminescence immunoassay led successfully to higher sensitivity in the PIVKA-II measurement. To realize higher sensitivity not only in the ECLIA, but also in an enzyme immunoassay, a chemiluminescence assay, a radioisotope assay, latex turbidimetry or the like, the influence of an unspecific reaction in a sample should be taken into consideration.
In the process of studies for eliminating the influence of the unspecific reaction in a sample in the PIVKA-II measurement, it has been found that sensitivity and specificity of the measurement could be improved by adding to the reagents, thrombin and/or an antibody that reacts with a sensitivity with human fibrin-like related substances. As the substances attributable to such an unspecific reaction in the sample, attention was directed first to fibrin or its related substances in the sample, and second to thrombin bound to fibrin or its related substances. In particular, when a polyclonal antibody is used as an anti-human prothrombin antibody for a second antibody or a labeled antibody, it may be subject to the interference of these unspecific reaction substances, thereby causing positive errors in measurement of PIVKA-II. It is reported that the protein structure of prothrombin is composed of an F1 fragment, an F2 fragment and thrombin. The labeled antibody used for measurement of PIVKA-II may be not only an anti-prothrombin antibody, but also an anti-F1 antibody, an anti-F2 antibody, or an anti-(F1+F2) antibody. However, in consideration of the purity of these antibodies or the similarity of thrombin to the antigen, these antibodies may also react with bound or free thrombin in a sample. Further, in measurement of PIVKA-II, fibrin or its insoluble related substances in a sample are physically adsorbed onto carriers such as magnetic beads, glass beads, latexes, plastic plates or the like to give rise to the phenomenon of positive errors in the measurement.
To prevent the interference attributable to fibrin-like related substances and thrombin, antibodies reacting with the human fibrin-like related substances, for example, anti-fibrinogen or anti-fibrin antibodies, and/or thrombin are added to the reagents. Thus, the present invention succeeds in accurately measuring a very small amount of PIVKA-II by effectively inhibiting the nonspecific reaction.
An object of the present invention is to provide an immunoassay utilizing an antigen-antibody reaction for specifically measuring with a high sensitivity PIVKA-II in serum or plasma by adding to reagents thrombin and/or an antibody reacting with human fibrin-like related substances to the reagents.